Tyrosine sulfation is a common post-translational modification in secreted and membrane-bound proteins (Kehoe and Bertozzi, “Tyrosine sulfation: a modulator of extracellular protein-protein interactions,” Chem Biol 7:R57-61 (2000)). Although we are only beginning to understand the extent of its biological function, sulfotyrosine has already been identified in several protein-protein interaction paradigms. For example, tyrosine sulfation plays a determining role in chemokine binding to the chemokine receptors CCR2 (Preobrazhensky et al., “Monocyte chemotactic protein-1 receptor CCR2B is a glycoprotein that has tyrosine sulfation in a conserved extracellular N-terminal region” J Immunol 165:5295-5303 (2000)), CCR5 (Farzan et al., “Tyrosine sulfation of the amino terminus of CCR5 facilitates HIV-1 entry” Cell 96:667-676 (1999)), CXCR4 (Farzan et al., “The role of post-translational modifications of the CXCR4 amino terminus in stromal-derived factor 1 alpha association and HIV-1 entry,” J Biol Chem 277:29484-29489 (2002); Veldkamp et al., “Recognition of a CXCR4 sulfotyrosine by the chemokine stromal cell-derived factor-1alpha (SDF-1alpha/CXCL12),” J Mol Biol 359:1400-1409 (2006)) and CX3CR1 (Fong et al., “CX3CR1 tyrosine sulfation enhances fractalkine-induced cell adhesion,” J Biol Chem 277:19418-19423 (2002)). Similarly, leukocyte rolling under hydrodynamic shear stresses requires sulfation of PSGL-1 for proper binding and adhesion (Somers et al., “Insights into the molecular basis of leukocyte tethering and rolling revealed by structures of P- and E-selectin bound to SLe(X) and PSGL-1,” Cell 103:467-479 (2000)). Tyrosine sulfation is also involved in the coagulation cascade, having been identified in several clotting factors as well as in natural thrombin inhibitors such as the leech-secreted anticoagulant hirudin (Dong et al., “Tyrosine sulfation of the glycoprotein Ib-IX complex: identification of sulfated residues and effect on ligand binding,” Biochemistry 33:13946-13953 (1994); Bagdy et al., “Hirudin,” Methods Enzymol 45:669-678 (1976)). In addition, it was recently discovered that tyrosine sulfation on an antibody variable loop region is responsible for the neutralizing activity of a subset of CD4-induced HIV-1 antibodies, thus demonstrating the ability of sulfotyrosine to augment antibody-antigen affinity (Choe et al., “Tyrosine sulfation of human antibodies contributes to recognition of the CCR5 binding region of HIV-1 gp120,” Cell 114:161-170 (2003); Xiang et al., “Functional mimicry of a human immunodeficiency virus type 1 coreceptor by a neutralizing monoclonal antibody,” J Virol 79:6068-6077 (2005)).
A major obstacle to determining the functions of sulfation in the over 60 known and over 2100 predicted proteins containing sulfotyrosine (based on a study of mouse protein sequences) is the ability to synthesize selectively sulfated proteins (Moore, “The biology and enzymology of protein tyrosine O-sulfation,” J Biol Chem 278:24243-24246 (2003)). Current methods rely on standard peptide synthesis or in vitro enzymatic sulfation (Veldkamp et al., “Recognition of a CXCR4 sulfotyrosine by the chemokine stromal cell-derived factor-1 alpha (SDF-1alpha/CXCL12),” J Mol Biol 359:1400-1409 (2006); Kirano et al., “Total synthesis of porcine cholecystokinin-33 (CCK-33),” J. Chem. Soc., Chem. Commun., 323-325 (1987); Muramatsu et al., “Enzymic O-sulfation of tyrosine residues in hirudins by sulfotransferase from Eubacterium A-44,” Eur J Biochem 223:243-248 (1994); Young and Kiessling, “A strategy for the synthesis of sulfated peptides,” Angew Chem Int Ed Engl 41:3449-3451 (2002)); however, both lack generality: the former is limited by length restrictions and the tendency towards sulfotyrosine desulfation under acidic conditions; the latter is limited by the availability of accessory sulfotransferases and their associated recognition sequence constraints.
The direct incorporation of a genetically encoded sulfotyrosine unnatural amino acid at defined sites in proteins directly in living organisms would overcome the limitations described above. The direct incorporation of sulfotyrosine will greatly facilitate the study of sulfation events in the regulation of biological processes and will also allow for the creation of sulfated antibody and peptide libraries of significant diversity. Furthermore, the ability to produce a sulfated form of the protein hirudin has immediate clinical application for use as an improved anticoagulant (improved relative to the non-sulfated form). What are needed in the art are new strategies for incorporation of sulfotyrosine unnatural amino acid into proteins.
A general methodology has been developed for the in vivo site-specific incorporation of diverse unnatural amino acids into proteins in both prokaryotic and eukaryotic organisms. These methods rely on orthogonal protein translation components that recognize a suitable selector codon to insert a desired unnatural amino acid at a defined position during polypeptide translation in vivo. These methods utilize an orthogonal tRNA (O-tRNA) that recognizes a selector codon, and where a corresponding specific orthogonal aminoacyl-tRNA synthetase (an O-RS) charges the O-tRNA with the unnatural amino acid. These components do not cross-react with any of the endogenous tRNAs, RSs, amino acids or codons in the host organism (i.e., it must be orthogonal). The use of such orthogonal tRNA-RS pairs has made it possible to genetically encode a large number of structurally diverse unnatural amino acids.
The practice of using orthogonal translation systems that are suitable for making proteins that comprise one or more unnatural amino acid is generally known in the art, as are the general methods for producing orthogonal translation systems. For example, see International Publication Numbers WO 2002/086075, entitled “METHODS AND COMPOSITION FOR THE PRODUCTION OF ORTHOGONAL tRNA-AMINOACYL-tRNA SYNTHETASE PAIRS;” WO 2002/085923, entitled “IN VIVO INCORPORATION OF UNNATURAL AMINO ACIDS;” WO 2004/094593, entitled “EXPANDING THE EUKARYOTIC GENETIC CODE;” WO 2005/019415, filed Jul. 7, 2004; WO 2005/007870, filed Jul. 7, 2004; WO 2005/007624, filed Jul. 7, 2004 and WO 2006/110182, filed Oct. 27, 2005, entitled “ORTHOGONAL TRANSLATION COMPONENTS FOR THE VIVO INCORPORATION OF UNNATURAL AMINO ACIDS.” Each of these applications is incorporated herein by reference in its entirety. For additional discussion of orthogonal translation systems that incorporate unnatural amino acids, and methods for their production and use, see also, Wang and Schultz, “Expanding the Genetic Code,” Chem. Commun. (Camb.) 1:1-11 (2002); Wang and Schultz “Expanding the Genetic Code,” Angewandte Chemie Int. Ed., 44(1):34-66 (2005); Xie and Schultz, “An Expanding Genetic Code,” Methods 36(3):227-238 (2005); Xie and Schultz, “Adding Amino Acids to the Genetic Repertoire,” Curr. Opinion in Chemical Biology 9(6):548-554 (2005); Wang et al., “Expanding the Genetic Code,” Annu. Rev. Biophys. Biomol. Struct., 35:225-249 (2006; epub Jan. 13, 2006); and Xie and Schultz, “A chemical toolkit for proteins—an expanded genetic code,” Nat. Rev. Mol. Cell Biol., 7(10):775-782 (2006; epub Aug. 23, 2006).
There is a need in the art for the development of orthogonal translation components that incorporate sulfotyrosine unnatural amino acid into proteins, where the unnatural amino acid can be incorporated at any defined position. The invention described herein fulfills these and other needs, as will be apparent upon review of the following disclosure.